Compositions and methods for improving resistance to breakage and eggshell strength

ABSTRACT

Described herein are FGF-23 epitope peptides, methods of producing antibodies in laying hens by injecting the peptides, and methods of improving resistance to eggshell breakage and/or increasing eggshell strength by administering an FGF-23 epitope peptide to a laying hen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application62/374,224 filed on Aug. 12, 2016, which is incorporated herein byreference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure is related to compositions and methods forreducing egg breakage and/or by improving the eggshells of eggs laid bylaying hens.

BACKGROUND

It has been estimated that the income loss to U.S. producers from brokeneggs is $266 million/year based on 240 million laying chickens. Anadditional loss of $211 million occurs due to uncollectable eggs atprocessing plants. Today, the U.S. has 338 million laying chickens, andegg loss due to breakage or uncollectable eggs could account for $375million and $297 million/year, respectively. Global hen numbers are 6.6billion and could account for losses due to breakage or uncollectableeggs of $7.3 billion and $5.8 billion respectively. With as much as 20%of the eggs lost from a given hen (300 egg/year), a total of $3.50 of anindividual hen's production is lost due to egg breakage. The eggspecific gravity, percent shell, and force to break the shell (grams offorce) are negatively correlated with egg breakage in commercial eggproduction and packing facilities, with specific gravity and percentshell having a correlation coefficient of ≧0.85 (C. F. Strong, 1989,Poultry Science 68:1730-1733). A strategy that would reduce egg loss dueto breakage would be of considerable value to egg producers.

There are no products that are currently marketed that consistentlyincrease egg specific gravity, eggshell amount and force to break eggs(measures of “eggshell strength”) and thereby reduce egg breakage. Forexample, while calcium and vitamin D are two critical nutrients forassuring proper calcification of eggs, these nutrients are currently fedat levels that maximize eggshell strength. Sodium bicarbonate canrestore shell strength caused by a depletion of blood bicarbonate whenenvironmental temperatures are high such as in the summer, however,sodium bicarbonate does not actually increase shell strength per se.

What is needed are compositions and methods for reducing breakage andincreasing eggshell strength in eggs laid by laying hens.

BRIEF SUMMARY

In an aspect, an FGF-23 epitope peptide has the sequence SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In an aspect, the FGF-23epitope peptide is conjugated to a carrier protein.

In another aspect, a pharmaceutical composition comprises apharmaceutically acceptable excipient and an FGF-23 epitope peptide,wherein the FGF-23 epitope peptide has the sequence SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20.

In an aspect, a method of improving resistance to eggshell breakageand/or improving eggshell strength comprises administering to a layinghen an effective amount of an FGF-23 epitope peptide to improveresistance to eggshell breakage and/or improve the eggshell strength ofeggs laid by the hen, wherein the FGF-23 epitope peptide consists of asequence that is not identical to a sequence in human FGF-23.

In another aspect, a method of producing antibodies in a laying hencomprises administering to the laying hen an effective amount of anFGF-23 epitope peptide, wherein the FGF-23 epitope peptide has thesequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing g of force need to break the eggshell foreggs collected from hens injected with control, NP1, or NP7 peptide.

FIG. 2 is a bar graph showing egg specific gravity for eggs collectedfrom hens injected with control, NP1, or NP7 peptide.

FIG. 3 is a bar graph showing shell weight (g) for eggs collected fromhens injected with control, NP1, or NP7 peptide.

FIG. 4 is a bar graph showing shell index (% of whole egg) for eggscollected from hens injected with control, NP1, or NP7 peptide.

FIG. 5 shows eggshell strength in g of force for eggs from 37 and 76week old hens treated with control or NP7 peptide.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

As described in U.S. Pat. No. 9,078,842, hens injected with an FGF-23epitope peptide have reduced excreta phosphate. In addition, the chicksof these hens have reduced phosphorus deficiency when fed lownon-phytate phosphorus diets. During subsequent studies, the presentinventors observed that the eggs from hens injected with this FGF-23epitope peptide were more resistant to breakage than control injectedhens. More specifically, eggs from hens injected with FGF-23 epitopepeptides GMNPPPYS (NP1; SEQ ID NO: 1) or YTSTERNSFH (NP7; SEQ ID NO: 3)had significantly greater (P<0.001) shell weight, percent shell weight,and specific gravity (a measure of shell quantity) than control injectedeggs. Other egg variables such as egg size, internal component content,and dry matter of egg internal components were not consistently affectedby vaccine type (FGF-23 epitope peptide or control). For example, toquantify the results, eggs from control and FGF-23 epitope peptidevaccinated hens were subjected to load resistance testing using a StableMicro System. Eggs from hens vaccinated with either FGF-23 epitopepeptide required 30% more force to break the egg (P<0.001) as well assignificant increased other measures of shell strength. Thus, thepresent inventors have unexpectedly found that administration of FGF-23epitope peptides to laying hens provides eggs with improved resistanceto shell breakage and/or improved shell strength, which will increasethe number of collectable (i.e., intact) eggs produced by the hens.

In one aspect, a FGF-23 epitope peptide comprises an epitope sequencefrom avian FGF-23. An FGF-23 epitope peptide does not include afull-length FGF-23 sequence, and typically contains 8-12, morespecifically 8-10 amino acid residues. In one embodiment, the avianFGF-23 epitope peptide consists of an epitope that is not found in humanFGF-23, such that the antibodies produced upon vaccination of a hen willnot cross-react with human FGF-23. The homology of the NP1 and NP7peptides between human and chicken is shown in Table 1:

TABLE 1 Homology of FGF-23 epitope between chicken and human NP1 NP7Human GMNPPPYS YTATARNSYH (SEQ ID NO: 1) (SEQ ID NO: 2) Chicken GMNPPPYSYTSTERNSFH (SEQ ID NO: 1) (SEQ ID NO: 3)

Exemplary avian FGF-23 epitope peptides are provided in Table 2:

TABLE 2 Avian FGF-23 epitope sequences SEQ ID Peptide SequenceDescription NO: NP-1 GMNPPPYS chicken 1 NP-7h YTATARNSYH human 2 NP-7YTSTERNSFH chicken, turkey, 3 quail NP-7D YTSSERNSFH duck 4 ESS-1LLNPSWGN chicken, duck, 5 turkey, quail ESS-2 NSSPLLNP chicken, duck, 6turkey, quail ESS-3 KSEGAGCV chicken, duck 7 ESS-3TQ KSEGAGSVturkey, quail 8 ESS-4 STERNSFH chicken, turkey, 9 quail ESS-4D SSERNSFHduck 10 ESS-5 HINGVPHQ chicken 11 ESS-5D YIDGVPHQ duck 12 ESS-5THISGVPYQ turkey 13 ESS-5Q HISGVPHQ quail 14 ESS-6 NTPEPHRNchicken, duck, 15 turkey, quail ESS-7 VPHQTIYS chicken, duck, quail 16ESS-7T VPYQTIYS turkey 17 ESS-8 ITGVKSGR chicken, duck, 18 turkey, quailESS-9 QINADGHI chicken, turkey, 19 quail ESS-9D QINADGYI duck 20

In one embodiment, the FGF-23 epitope peptide comprises, consistsessentially of, or consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In another aspect, the FGF-23epitope peptide comprises, consists essentially of, or consists of SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

In one embodiment, the FGF-23 epitope peptide is conjugated to a carriersuch as a carrier protein, for example, for formulation into a vaccine.Exemplary carrier proteins include bovine gamma globulin, bovine serumalbumin, keyhole limpet hemocyanin, ovalbumin, or a protein that, whenconjugated with the peptide, elicits an antibody to the attachedpeptide.

The epitope peptide may be conjugated (e.g., covalently conjugated) tothe carrier protein according to methods known in the art. In someembodiments, the epitope sequence is conjugated to the carrier proteinvia a glutaraldehyde linking moiety. In other embodiments, the epitopesequence is conjugated to the carrier protein via maleimide mediatedconjugation. In some embodiments, the epitope sequence is conjugated via1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)mediated conjugation. Other methods of hatpen-carrier proteinconjugation may be used and the method of conjugation is not critical aslong as antibodies to the desired conjugated peptide may be effectivelygenerated.

Also includes herein are pharmaceutical compositions, such as oral,aerosol and parenteral compositions, and more particularly vaccinecompositions, comprising an FGF-23 epitope peptide and apharmaceutically acceptable carrier.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the FGF-23 epitope peptide, optionally conjugatedto a carrier protein, together with a pharmaceutically acceptableexcipient, such as diluents, preservatives, solubilizers, emulsifiers,and adjuvants. As used herein “pharmaceutically acceptable excipients”are well known to those skilled in the art.

In an aspect, the FGF-23 epitope peptide is conjugated to a carrier foraerosol or oral administration. For example, the peptide can beconjugated to a virus or a particle such as a nanoparticle.Alternatively, the peptide can be encoded by a polynucleotide, wherein asequence encoding the peptide is operably linked to expression controlsequences as are known in the art, such that upon administration to ahost, the peptide is synthesized, allowing for production of antibodiesthat neutralize FGF-23 in the host.

For oral administration, a pharmaceutical composition can take the formof, for example, a tablets or a capsule prepared by conventional meanswith a pharmaceutically acceptable excipient. Liquid preparations fororal administration can take the form of, for example, solutions,syrups, or suspensions, or they can be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations can be prepared by conventional means withpharmaceutically acceptable additives.

Aerosol formulations can be administered via inhalation and can bepropellant or non-propellant based. For example, embodiments of thepharmaceutical formulations of the disclosure comprise a peptide of thedisclosure formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like. Foradministration by inhalation, the compounds can be delivered in the formof an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer A non-limiting example of a non-propellant is a pump spraythat is ejected from a closed container by means of mechanical force(i.e., pushing down a piston with one's finger or by compression of thecontainer, such as by a compressive force applied to the container wallor an elastic force exerted by the wall itself (e.g., by an elasticbladder)).

In an aspect, the FGF-23 epitope peptide is administered parenterally ina sterile medium, either subcutaneously, intravenously, intradermally,intraperitoneally, or intramuscularly. Depending on the vehicle andconcentration used, the peptide can either be suspended or dissolved inthe vehicle. Advantageously, adjuvants such as a local anaesthetic,preservative and buffering agent can be dissolved in the vehicle mayalso be administered parenterally in a sterile medium, eithersubcutaneously, or intravenously, or intradermally, orintraperitoneally, or intramuscularly, in the form of sterile injectableaqueous or oleaginous suspensions. Depending on the vehicle andconcentration used, the peptide can either be suspended or dissolved inthe vehicle. Advantageously, adjuvants such as a local anaesthetic,preservative and buffering agent can be dissolved in the vehicle.

A vaccine composition may comprise an immunopotentiator as thepharmaceutically acceptable excipient, specifically an immunologicaladjuvant. Exemplary adjuvants include individually or mixtures of alum,aluminum phosphate, aluminum hydroxide, C-phosphae guanosine (CPG),squalene, and oil-based adjuvants including Freund's Complete andIncomplete adjuvant or others as listed in Cooper, P. D. (The selectiveinduction of different immune responses by vaccine adjuvants; Strategiesin Vaccine Design. G. L. Ada, ed. R. G. Landes Company, Austin. Tex.(1994)). A specific adjuvant is Freund's complete adjuvant. Anotherspecific adjuvant is Freund's incomplete adjuvant. Branded commercialadjuvants are also available and are suitable such as, for example,TiterMax®, AddaVax™, and Alhydrogel®. Exemplary concentrations of theFGF-23 epitope peptide in an adjuvant formulation generally are about 50micrograms to about 6 milligrams, specifically about 50 micrograms toabout 3 milligrams, even more specifically between about 50 microgramsto about 1 milligram.

In an aspect, a method of producing antibodies in a laying hen comprisesadministering (e.g., injecting) to the laying hen an effective amount ofan FGF-23 epitope peptide, wherein the FGF-23 epitope peptide has thesequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. Exemplary hens include chicken, turkey, duck, goose, peafowl, emu,pheasant, guinea fowl, quail, and the like. Administering the FGF-23epitope peptide provides FGF-23 neutralizing antibodies, that is,antibodies produced during an immune response in the hen that neutralizeFGF-23 that she produces. Since eggs are consumed by humans, in anaspect the antibodies will not neutralized FGF-23 in the consumer of theeggs, since the peptide injected into the chickens is not homologouswith the human peptide sequence.

In one aspect, a method of improving resistance to eggshell breakageand/or improving eggshell strength comprises administering (e.g.,intramuscular injection) a laying hen with an effective amount of anFGF-23 epitope peptide to improve resistance to eggshell breakage and/orimprove eggshell strength. Improving the resistance to breakage and/oreggshell strength will provide improve the number of collectable eggsproduced by the hen. In one embodiment, the FGF-23 epitope peptideconsists of a sequence that is not identical to a sequence in humanFGF-23. In another embodiment, the method comprises injecting a layinghen with a peptide of any one of SEQ ID Nos. 1-20. In anotherembodiment, the method comprises injecting a laying hen with a peptideof any one of SEQ ID Nos. 3-20. Exemplary hens include chicken, turkey,duck, goose, peafowl, emu, pheasant, guinea fowl, quail, and the like.Injection of the hens with the FGF-23 epitope peptide provides maternalFGF-23 neutralizing antibodies, that is, antibodies produced during animmune response in the hen that inhibit the action of maternal FGF-23.

In an aspect, the laying hen is greater than 35 weeks in age, andadministration prevents eggshell deterioration as the laying hen ages.

As used herein, the phrases “improve resistance to eggshell breakage”and “improve eggshell strength” of an egg means that, on average, thegrams of force needed to break the egg shell is increased by greaterthan or equal to 10%, 15%, 20% or more compared to eggs laid by similarhens that are not injected with an FGF-23 epitope peptide. Shellstrength can be measured by any one or more of load resistance testingwith a 50-kg load cell, 2 mm/s test speed, 0.001 kg trigger force, and a50 mm diameter cylindrical probe; egg shell specific gravity measured byflotation in sodium chloride and water solutions having sodium chlorideconcentrations of 1.070, 1.074, 1.078, 1.082, 1.086, 1.090, 1.094, and1.098M; or shell weight.

According to some methods of the present disclosure, a laying hen isadministered a composition comprising an FGF-23 epitope peptide, forexample an FGF-23 epitope peptide conjugated to a carrier protein. Thecomposition, e.g., the conjugate vaccine, may be carried in an adjuvant,e.g., Freund's complete adjuvant. In some embodiments, the laying henmay be vaccinated in combination with a conventional vaccinationregimen. Administration may occur once or multiple times, e.g., asecondary booster administration about one to two weeks after theprimary administration. A secondary booster administration may comprisethe same adjuvant or a different adjuvant, e.g., Freund's incompleteadjuvant.

Administration of the peptide elicits an immune system response, whichresults in the production of antibodies against an endogenous protein,specifically, antibodies against endogenous FGF-23. The laying hen'simmune system recognizes the epitopes, which mobilizes the preparationof maternal antibodies that recognize the FGF-23 epitope peptidesequence.

The invention is further illustrated by the following non-limitingexamples.

Example 1: Effect of Anti-FGF-23 Antibodies on Egg Quality of LayingHens Methods

Peptide Conjugation and Hen Vaccination: Two synthetic chicken FGF-23epitope peptides (NP1, GMNPPPYS (SEQ ID NO: 1); and NP7, YTSTERNSFH (SEQID NO: 3); synthesized by GeneScript, Piscataway, N.J.) wereindividually conjugated to bovine gamma globulin (BgG, Sigma, St. Louis,Mo.) using a glutaraldehyde conjugation procedure. Briefly, 10 mg of BgGand 10 mg peptide were dissolved in 2 mL of 0.1 M sodium acetate buffer(PH=7). 1.3 ml of 2 M glutaraldehyde was then added to the buffer,followed by a 3 hour conjugation period. Peptide conjugation was thenstopped by adding 100 mg glycine in the reaction system for 1 hour. Theconjugation solution was dialyzed against PBS (PH=7) overnight indialysis bag with a cutoff molecular weight of 6000-8000. For primaryvaccination, FGF-23 epitope peptide-BgG conjugates (dialysate) wereprepared as vaccines using Freund's Complete Adjuvant as the adjuvantand intramuscularly injected (breast and thigh muscle) into 30 SingleComb White Leghorn laying hens (15 hens for NP1-BgG conjugate, and 15hens for NP7-BgG conjugate). For booster injections, FGF-23 peptide-BgGconjugates (dialysate) were prepared as vaccines using Freund'sIncomplete Adjuvant as the adjuvant. Four booster injections were givenafter 2 weeks, 4 weeks, 24 weeks and 44 weeks following the primaryinjection to maintain antibody levels. Another 14 hens were injectedwith unconjugated BgG (no peptide conjugate) using the same time pointsand adjuvants as described for the FGF-23 epitope peptide and served asthe control vaccinated hens. All the hens used were individually housedin cages with raised wire floors and maintained under a 16-hour light:8-hour dark lighting regimen with free access to feed and water.

Sample Collection: Eggs were collected for 20 consecutive days after 1week following the fourth booster injection. Egg weight (g), specificgravity (see below for analysis details), shell weight (g), yolk weight(g), albumen weight (g), dry matter content of the whole egg (%), drymatter content of the shell (%), dry matter content of the yolk (%), anddry matter content of the albumen (%) were determined. Also calculatedwere shell index (shell weight/egg weight×100%), yolk index (yolkweight/egg weight×100%), albumen index (albumen weight/egg weight×100%),the percent of shell dry matter in whole egg dry matter (%), the percentof yolk dry matter in whole egg dry matter (%), the percent of albumendry matter in whole egg dry matter (%), and the yolk to albumen ratio(yolk weight:albumen weight, g:g) were calculated.

Egg specific Gravity: Solutions containing sodium chloride and waterwere created with molarity concentrations of 1.070, 1.074, 1.078, 1.082,1.086, 1.090, 1.094, and 1.098 at volumes of 1000 mL. The eggs wereindividually tested starting from lowest molarity to highest molarity,with the molarity in which the egg began floating recorded for thespecific gravity measurement.

Egg Shell Strength: For shell strength determination, eggs werecollected for 5 consecutive days after 4 weeks following the fourthbooster injection. Shell strength was determined using a TA.HDPlusTexture Analyzer (Stable Micro Systems, Texture Technologies Corp.,South Hamilton, Mass.) and Texture Expert software (Exponent Lite6,1,4,0). Shell strength determinations were conducted utilizing a 50-kgload cell, 5 mm/s pre-test speed, 2 mm/s test speed, 0.001 kg triggerforce, 50 mm diameter aluminum cylindrical probe (P/50), and egg holder(diameter 32 mm, height 27 mm, plastic cap).

Results

Hens were vaccinated with either Control (FCA, n=15), NP1 peptide(GMNPPPYS, n=15, SEQ ID NO: 1), or NP7 peptide (YTSTERNSFH, n=15; SEQ IDNO: 2). Eggs were collected during peak antibody production (within 2months after booster vaccination, when hens were 69 to 72 weeks of age).

TABLE 3 Effect of anti-FGF-23 antibodies on egg quality of 69- to71-wk-old laying hens FCA NP1 NP7 P-value (independent t-test) mean SEmean SE mean SE FCA&NP1 FCA&NP7 Shell Strength (g of force) 3093 1173995 135 4153 99 <0.001 <0.001 Specific Gravity 1.080 0.0004 1.0820.0004 1.083 0.0004 <0.001 <0.001 Shell Weight (g) 7.02 0.04 7.43 0.057.36 0.04 <0.001 <0.001 Shell Index (%) 12.95 0.07 13.49 0.07 13.34 0.07<0.001 <0.001 Egg Weight (g) 54.25 0.26 55.16 0.26 55.30 0.30 0.0140.080 Yolk Weight (g) 17.38 0.10 17.72 0.09 17.37 0.12 0.014 0.912 YolkIndex (%) 32.08 0.16 32.19 0.15 31.46 0.19 0.623 0.011 Albumen Weight(g) 29.30 0.21 29.46 0.22 31.46 0.24 0.595 0.035 Albumen Index (%) 53.940.22 53.31 0.23 54.12 0.24 0.050 0.583 Total Egg DM (%) 35.72 1.57 35.771.26 35.22 0.74 0.981 0.772 Yolk DM (%) 19.90 2.71 22.12 2.76 19.28 2.580.571 0.869 Shell DM (%) 74.00 1.18 75.47 1.96 75.13 2.71 0.526 0.705Albumen DM (%) 47.06 3.88 42.83 3.61 47.21 3.63 0.430 0.978 Yolk DMPercent in Total Egg DM (%) 27.55 2.63 30.42 2.91 27.70 2.82 0.470 0.970Shell DM Percent in Total Egg DM (%) 28.64 0.92 29.12 0.74 29.41 0.530.685 0.475 Albumen DM Percent in Total Egg DM 43.80 2.47 40.46 2.7242.89 2.75 0.370 0.806 (%) Yolk to Albumen Ratio (g/g) 0.60 0.01 0.610.01 0.59 0.01 0.206 0.189

As shown in FIGS. 1-4, the egg shell strength, measured as g of force,specific gravity of the eggs, shell weight in g and shell index,increased after injection of either NP1 or NP7 FGF-23 epitope peptide(when hens were 72-wk-old). Table 1 demonstrates that the presence ofantibody to FGF-23 had no adverse effects on any measure of internalquality; hence eggshell strength could be increased without adverselyaffecting internal or edible egg quality. The slight increase in eggweight in eggs from hens injected with an FGF-23 peptide was likely dueto the significant increase in eggshell weight. All other changes ininternal egg measurements were not consistent across the FGF-23 peptidesused. Most important is that yolk and albumen dry matter were notadversely affected by the use of FGF-23 peptide immunizations.Maintenance of dry matter is important to egg breaking operations whichwant to maintain solids levels for egg drying. The finding that apeptide (e.g., NP7) was as effective as NP1 in inducing maternalantibodies that increased shell strength was important. NP1 has sequencehomology to human FGF-23. Antibodies made the NP1 could possibly bindhuman FGF-23 if the egg containing the antibody was consumed by humansintact (not denatured through cooking) and the hormone was present inthe gastrointestinal tract lumen (FGF-23 has not been shown to bepresent in the gastrointestinal tract lumen or to have any biologicalrole in the gastrointestinal tract lumen). The potential crossreactivity of FGF-23 NP1 antibody to human FGF-23 could be viewed as apotential food safety issue. However NP7, which is much less homologousto the sequence in humans (3 nonmatching amino acids), suggest thatantibody to chicken FGF-23 (NP7) is less likely to cross react withhuman FGF-23.

Example 2: Effect of Anti-FGF-23 Antibodies on Egg Quality of YoungLaying Hens

Hens were vaccinated with either control or FGF-23 peptide vaccines atthe age of 26 weeks. Two booster injections were conducted 2 and 4 wkafter the primary injection. Hens were at peak antibody titer 1 wk afterthe second booster injection. Eggs were collected for 28 consecutivedays beginning when hens were at 31 weeks of age and when the antibodywas at maximum levels. Shell strength (the peak force value at shellbreakage) was determined using a TA.HDPlus Texture Analyzer (StableMicro Systems, Texture Technologies Corp., South Hamilton, Mass.) withTexture Expert software (Exponent Lite 6,1,4,0) at the University ofWisconsin-Madison Meat Science & Muscle Biology Laboratory. Shellstrength determinations were conducted utilizing a 50-kg load cell with5 mm/s pre-test speed, 2 mm/s test speed, 15 mm test distance, 0.001 kgtrigger force, a 50-mm diameter aluminum cylindrical probe (P/50), andan egg holder (diameter 32 mm, height 27 mm, plastic cap, eggs werepositioned blunt end up).

TABLE 4 Egg shell strength of FGF-23 vaccinated young laying hens (from31 to 35 wk of age) SEQ Number Shell Strength Treatment ID NO: of eggs(g of force) SE P-value* Control 99 4822 65 — NP1 1 97 4632 66 0.041ESS1 5 103 4799 82 0.832 ESS2 6 91 4765 72 0.559 ESS3 7 87 4380 73<0.001 ESS4 9 78 4322 72 <0.001 ESS5 11 96 4415 77 <0.001 ESS6 15 444762 121 0.635 ESS7 16 89 4281 73 <0.001 ESS8 18 97 4732 78 0.379 ESS919 97 4582 72 0.014 *Two tailed independent t-test against to thecontrol group.

As shown in Table 4, from this trial it was concluded that the FGF-23vaccine did not improve eggshell strength in the young (less than 35weeks of age) hens, while the FGF-23 vaccine did improve eggshellstrength in the older hens. It is well known that young hens producesmaller eggs with the same amount of eggshell (calcium carbonate) asolder hens. As the hen gets older the egg size increases and the eggshell is thinned to cover the same amount of surface area as the eggsfrom the older hens. The FGF-23 vaccine appears to protect egg shelldeterioration with hen aging.

Example 3: Effect of Anti-FGF-23 Antibodies on Egg Quality of Young andOld Laying Hens

Young (n=20, 32-wk-old) and old (n=20, 71-wk-old) laying hens wereinjected with either a control vaccine or FGF-23 peptide NP-7 vaccine(see Table 5). Two booster injections were conducted 2 and 4 wk afterthe primary injection. Hens were at peak antibody titer 1 wk after thesecond booster injection. The eggs were collected for 5 consecutive days(hens were 37 and 76 wk of age, respectively). Shell strength of theeggs were analyzed at UW-Meat Science & Muscle Biology Laboratory. Bloodsamples of these hens will be collected and analyzed for calcium,phosphate, FGF-23 and 1,25(OH)₂D₃ levels.

TABLE 5 Experimental design Hens Vaccine 32-wk-old Control (n = 10) NP-7(n = 10) 71-wk-old Control (n = 10) NP-7 (n = 10)

As shown on FIG. 5. FGF-23 peptide NP-7 vaccine increased eggshellstrength of eggs from 37- and 76-wk-old laying hens. These anticipatedresults may be explained by plasma levels of phosphate, calcium, FGF-23and 1,25(OH)₂D₃.

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second etc.as used herein are not meant to denote any particular ordering, butsimply for convenience to denote a plurality of, for example, layers.The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

1. An FGF-23 epitope peptide, wherein the peptide has the sequence SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
 7. SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, or SEQ ID NO:
 20. 2. The FGF-23 epitopepeptide of claim 1, wherein the FGF-23 epitope peptide is conjugated toa carrier protein.
 3. The FGF-23 epitope peptide of claim 2, wherein thecarrier protein is bovine gamma globulin, bovine serum albumin, keyholelimpet hemocyanin, or ovalbumin.
 4. A pharmaceutical compositioncomprising a pharmaceutically acceptable excipient and an FGF-23 epitopepeptide, wherein the FGF-23 epitope peptide has the sequence SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
 7. SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO:
 20. 5. The pharmaceuticalcomposition of claim 4, wherein the FGF-23 epitope peptide is conjugatedto a carrier protein.
 6. The composition of claim 5, wherein the carrierprotein is bovine gamma globulin, bovine serum albumin, keyhole limpethemocyanin, or ovalbumin.
 7. The pharmaceutical composition of claim 5,wherein the composition is for oral, aerosol, or parenteraladministration.
 8. The pharmaceutical composition of claim 7, whereinthe pharmaceutically acceptable excipient is an immunological adjuvant.9. A method of improving resistance to egg shell breakage and/orimproving egg shell strength, comprising administering to a laying henan effective amount of an FGF-23 epitope peptide to improve resistanceto egg shell breakage and/or improve the egg shell strength of eggs laidby the hen, wherein the FGF-23 epitope peptide consists of a sequencethat is not identical to a sequence in human FGF-23.
 10. The method ofclaim 9, wherein the FGF-23 epitope peptide has the sequence SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
 7. SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO:
 20. 11. The method of claim 9,wherein the laying hen is greater than 35 weeks in age, andadministration prevents eggshell deterioration as the laying hen ages.12. The method of claim 9, further comprising administering to thelaying hen a booster administration one or more weeks or more after thefirst administration, wherein the booster administration comprises aneffective amount of an FGF-23 epitope peptide to improve resistance toegg shell breakage and/or improve the egg shell strength of eggs laid bythe hen, wherein the FGF-23 epitope peptide has the sequence SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
 7. SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO:
 20. 13. The method of claim 9,wherein the FGF-23 epitope peptide is conjugated to a carrier protein.14. The method of claim 12, wherein the carrier protein is bovine gammaglobulin, bovine serum albumin, keyhole limpet hemocyanin, or ovalbumin.15. The method of claim 9, wherein the laying hen is a chicken, aturkey, a duck, a goose, a peafowl, an emu, a pheasant, a guinea fowl,or a quail.
 16. The method of claim 9, wherein administering is oral,aerosol, or parenteral administration.
 17. The method of claim 9,wherein improved resistance to egg shell breakage and/or improved eggshell strength is determine by one or more of: load resistance testingwith a 50-kg load cell, 2 mm/s test speed, 0.001 kg trigger force, and a50 mm diameter cylindrical probe; egg specific gravity measured byflotation in sodium chloride and water solutions having sodium chlorideconcentrations of 1.070, 1.074, 1.078, 1.082, 1.086, 1.090, 1.094, and1.098M; and shell weight.
 18. A method of producing antibodies in alaying hen, comprising administering to the laying hen an effectiveamount of an FGF-23 epitope peptide, wherein the FGF-23 epitope peptidehas the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO:
 7. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:
 20. 19.The method of claim 18, wherein the FGF-23 epitope peptide is conjugatedto a carrier protein.
 20. The method of claim 19, wherein the carrierprotein is bovine gamma globulin, bovine serum albumin, keyhole limpethemocyanin, or ovalbumin.
 21. The method of claim 18, wherein the layinghen is a chicken, a turkey, a duck, a goose, a peafowl, an emu, apheasant, a guinea fowl, or a quail.
 22. The method of claim 18, whereinadministering is intramuscular injection.